Xeronine, a new alkaloid, useful in medical, food and industrial fields

ABSTRACT

Described herein are the composition, the characterization, the assay, the mode of action and the utility of a new alkaloid which may be isolated from a wide variety of natural materials by observing certain techniques and precautions herein set forth.

This is a continuation-in-part of application Ser. No. 870,919, filedJan. 19, 1978, now abandoned.

BACKGROUND

Certain natural products, such as various enzymes, herbs, and plantextractives are used in medicine, food technology, and industry toperform critical functions. For example a range of "crude enzyme"products are used as the basis for preparing oral antiinflammatorydrugs. Bromelain, papain, amylase, pancreatin, chymotrypsin, a proteasefrom Serratia marcescens and egg white lysozyme are all occasionallyeffective as oral antiinflammatory agents. Unfortunately even though allof these enzymes may be rigidly standardized for the named enzyme, thepharmacological effectiveness of different batches of these enzymesvaries greatly. Obviously all of the members of this wide range ofenzymes contains some pharmacologically active ingredient other than thenamed enzyme. If the nature and composition of this unknown ingredientcould be identified and standardized, then preparations could be madewhich would be more effective than the improperly standardized presentproducts and new applications could be developed, since the new productwould be reliable. This is one of the objectives of the presentinvention.

In food technology three of the most important applications of "enzymes"are the "chillproofing" of beer, the in vivo and in vitro tenderizationof meat, and the preparation of "instant" cooking cereals. Althoughproteases are used in all of these applications, in no case is there anycorrelation between proteolytical activity and effectiveness of thepreparation. Swift & Co. after much research on their "proten" processfound that no known enzyme test could distinguish between effective andnon-effective batches of enzymes. On the whole they found that the mostproteolytically active samples of papain were the least effective meattenderizing agents. Since each assay on living animals cost them (1963figures) $25,000, the importance of this problem can readily beappreciated. The Cream of Wheat Co. in a patented process attempted touse proteases to prepare "instant cooking" cereal. They had so muchdifficulty in getting reproducible results, even though they purchasedenzymes having exactly the same specifications from the same company,that for many years they discontinued using the process. Formulators ofbeer "chill-proofing" preparations have long realized that each newbatch of enzyme which they bought was a gamble. They have never beenable to formulate their products on the basis of "proteolytic activity."Instead the better formulators all rely solely upon laboratory use-testsin which the batches of enzymes are evaluated on the basis of their"chill-proofing" ability. If a simple, meaningful assay could be devisedwhich would give a proper evaluation of performance, their costs wouldbe greatly reduced. There have also been some interesting potentialapplication of "enzymes" which have never developed commercially sinceone batch of "enzyme" might perform satisfactorily whereas the nextbatch from the same company and having precisely the same specificationswould be worthless. The production of better flavored cocoa and vanillaare two examples of such applications.

Three things convinced me that the primary active ingredient in allthese "enzymes" was something other than an enzyme; (1) the enzymerationale for pharmacological activity was completely incompatible withaccepted physiological and anatomical data, (2) both clinical andlaboratory data showed a lack of correlation between enzymatic activity,and pharmacological action, and (3) certain (but not all) boiled enzymepreparations were as active as were the unheated enzyme preparations.Although certain clues quickly suggested to me that the activeingredient was a small molecule rather than a protein or largepolypeptide, working out the details by which this small molecule isproduced proved to be a difficult problem. The final solution of thisproblem now makes it possible to both describe useful methods forproducing products containing the active ingredient and to suggest manynew combinations and applications for the future.

RELATIONSHIP OF THE PRESENT INVENTION TO PREVIOUS KNOWLEDGE ANDPRACTICES

The present invention differs from all previous practices and knowledgeof the use of oral antiinflammatory enzyme as well as certain branchesof folklore medicine which also treat inflammation, high blood pressure,and many other ailments in that this invention identifies the activeingredient, describes how the active ingredient is produced in the plantor animal, gives directions for liberating and isolating the activeingredient or its precursor, develops a theory of how the activeingredient works in pharmacological, physiological, food and industrialapplications, and suggests and illustrates new applications for theactive ingredient.

By contrast in the herbal folklore field, either no attempt is made toidentify the active ingredient or else the active ingredient isincorrectly identified. Thus although many investigators haveinvestigated the active ingredient in genseng, the very fact that everyother year additional materials are isolated and claimed to be theactive ingredient shows that the true nature of the active ingredientcontinues to elude the investigators. Perhaps the most eggregiousexample of a misidentification of an active ingredient occurs with"Laetrile," a reputed cure for cancer. The unfortunate designation ofamygdalin as the active ingredient of "Laetrile" has caused the waste ofmillion of dollars both on research and on futile use of the material. Afew investigators have correctly pointed out that perhaps the wrongmaterial was being isolated from almonds. However, these investigatorshave been unable to identify the nature of the active ingredient whichis occasionally accidentally included in the product.

Another classical example of the misidentification of the activeingredient in a potentially valuable product is the reputedidentification of proteases as the pharmacologically active ingredientin bromelain, pancreatin, chymotrypsin, and Serratia marcescensprotease. Thus, since these products are incorrectly standardized tocontain identical amounts of protease activity the biological activityvaries greatly between different batches of product. For example Dr.Klein found that different batches of bromelain varied greatly in theirability to remove burn eschars, (incidentally this is one of the fewmedical applications of a protease which theoretically appears to bejustified. However, cooperative work which Dr. Klein and I did showedthat the protease activity was unrelated to the debridement of burneschars.) Dr. G. Gerard of France showed that different batches ofbromelain varied in their ability to cure certain types of cancer. Thusthis potentially valuable remedy could never be recommended as a cure.

The only published work which makes a new suggestion for a specificingredient for bromelain is that of Klein and Houck, U.S. Pat. No.4,197,291. Klein and Houck isolated and described a non-proteolytichydrolytic enzyme which they believe is the active ingredient ofbromelain. However, these authors were neither able to identify thenature of the substrate for this enzyme nor could they describe what theenzyme did at the molecular level. Although these authors call theirenzyme a hydrolytic enzyme, the only evidence which they cite is theclinical evidence that the enzyme promotes the debridement of burneschars by a collagenase type action. Their data on the size of theenzyme and its dimer or trimer nature are excellent.

The enzyme which these authors have isolated appears to be identical tothe one which I had shown was necessary for the liberation of the activeingredient in 1972. Since I was searching for the ultimate moleculewhich was responsible for the pharmacological activity, I merelyconsidered the enzyme as one part of several components which werenecessary to finally liberate the active substance. Without a propersubstrate, the enzyme is worthless. My personal belief, which is basedupon my discovering that xeronine is biologically active down toconcentrations of 10⁻¹⁰ g/g of tissue, is that the enzyme which theyisolated was contaminated by xeronine and that the adsorbed xeronine,not the enzyme, was responsible for the biological activity which theyobserved. Thus their product would--similar to bromelain--bestandardized on the basis of the incorrect ingredient.

A similar situation occurs with chymotrypsin, a widely used oralantiinflammatory agent. Since chymotrypsin is isolated from the pancreasand since I had previously found that certain batches of pancreatincontained xeronine, I felt that trace amounts of xeronine might havebeen adsorbed on the protein during the isolation procedures. Afterheating a solution of chymotrypsin to 85 C. for five minutes, I foundthat the solution contained no protease activity but still contained 3CAU of xeronine activity per mg, a figure similar to that of manybromelain samples.

From these examples--any many more could be cited--it is readilyapparent that in the areas mentioned, the present practices andknowledge are inadequate to solve the problems for which the productsare being recommended. My invention will greatly advance the treatmentof certain medical problems, the modification of certain food products,and the recovery of certain industrial waste products by providingpreparations which are standardized to contain the active ingredient forthese various applications.

BRIEF DESCRIPTION OF THE DRAWINGS

The FIGURE shows fractionation of the biological activity of arelatively pure sample of xeronine.

DETAILED DESCRIPTION OF THE INVENTION

XERONINE, Chemistry

Alkaloid Nature: The alkaloid nature of xeronine is attested to by themethod used for the isolation and purification of the material, by itschanges in the UV spectrum when it is heated, by its behaviour in a massspectrum analysis instrument and by its chemical reactions. In additionits odor is very similar to other alkaloid materials and is sufficientlydistinctive so that once a person has been exposed to the odor, he canreadily recognize the odor in other preparations.

During the isolation and purification procedures, the active ingredientbehaves as a classical base. At pH values above 7.8 it can readily beextracted from aqueous solutions into such organic solvents aschloroform, ethylacetate, ether, or butanol. By extracting the organiclayer with weak acid solutions, the alkaloid can be taken up in theaqueous phase.

This phase transfer procedure should be repeated several times. Duringthe initial extraction into the organic solvent layer, the base isaccompanied by steroids and camphor. Apparently xeronine can associatequite firmly with these lipids with the result that in the firstextraction of the base from the organic layer, some of the xeronineremains in the organic layer with the lipids and some of the neutrallipds are extracted into the acidic water phase with the xeronine.However, after several reextractions the separation is clean.

Although the isolation procedures prove that the active ingredient is abase, it does not provide any clues regarding the nature of the base.When the active ingredient is isolated by gentle techniques, that iswithout exposure to high or low pH values or heat, it contains no freeamino groups as measured by the ninhydrin reaction. However, if thematerial is heated strongly on a chromatographic plate, it finallyreacts with ninhydrin to give an organish pink color which is nottypical of a free amine. If, on the other hand, the active ingredient isheated with acid or base, then the material gives a typical ninhydrincolor. These reactions prove that the nitrogen is originally presenteither in a secondary or tertiary form.

The changes which occur when the active ingredient is heated underdifferent pH conditions give the most valuable information about thenature of the molecule. If the material is heated at pH 1.5-2.0 at about60 C. for about 24-48 hours, the original essentially flat UV spectrumdevelops a very sharp and intense peak at 280 nm. On the other hand if asolution of the active ingredient is heated at pH 9.5 at about 80-90 C.for ten minutes a very strong peak develops at 253 nm. If the pH ofeither the original solution, or the 280 nm solution, or the 253 nmsolution is adjusted to pH 12 and the solution is warmed slightly a newsharp peak appears at 245 nm and the 253 and the 280 nm peaks disappearcompletely.

The lack of any UV absorption except at low wavelengths indicates amolecule which contains no double bonds or other UV chromophores. Theformation of forms of xeronine which absorb strongly at 280, 253 or 245nm suggest dehydration of hydroxyl groups attached to non-aromatic ringswith the formation of double bonds. The interconversion of three formsof the molecule into a fourth form, namely the one absorbing at 245 nmindicates that the molecule must have several OH groups on severaldifferent non-aromatic rings. At different pH values dehydration occursat different parts of the rings. Apparently the 245 nm is the moststable form since it cannot be reconverted to the other forms. The 245nm form has no biological activity but is valuable as a potential assaytool.

Although all attempts to obtain useful mass spectral data on the activesubstance have been unsuccessful so far, even the lack of definitivedata supports the picture of a fairly complex alkaloid. Since thealkaloid is volatile enough to give a definite odor in the free baseform, one would expect that there would be no problem in getting adefinitive mass spectrum analysis of the product. Yet all of the testswhich have been run so far indicate that the molecule is so labile, thatwhen it is bombarded by electrons, to form the molecular ion, themolecule fragments into a wide range of break down products. With oneparticularly good sample which was obtained by distilling the alkaloidfrom a basic aqueous solution at pH 9, and then picking up with a needlethe crystals which float on the surface of the liquid at 30 and 50 C.two small peaks were obtained which had a mass of 414 and 428. At highertemperatures these peaks were part of a complex spectrum of productswhich continued to a mass of 518. Further data are required to get adefinitive mass for the molecule. However, temporarily I am assumingthat the molecular weight is 428, that the 414 peak represents the lossof nitrogen (although this is difficult to explain) and that the heaviermass peaks at higher temperatures represent aggregations of fragments ofthe molecule. As a minimum at this stage of the study we can feelconfident that the molecular weight must be between 413 and 518.However, since the material has a definite odor at room temperature, Ibelieve that the 428 mass may be the correct one.

The data from gel filtration would be completely compatible with amolecular weight of 428. When preparations of the active factor grosslycontaminated with salts, sugars, peptides and amino acids are run on agel filtration column (Sephadex G-15), the biological activity appearsafter the simple salts and sugars. This indicates a molecule which issmall enough to enter the pores of the gel but is large enough so thatwhen it diffuses out the pores it diffuses out after the simple sugarsand amino acids. The UV spectrum of this fraction shows that it containsa mixture of presumably the 253 nm form of the active substance andpeptides. The peptides were identified by the ninhydrin reaction.

Assay of Xeronine

Spectrographic Method: This method is based on the irreversibleconversion of the biologically active forms of xeronine to a formabsorbing strongly at 245 nm by warming a solution containing xeronineat pH 12, dropping the pH between 3 and 7 and then comparing thespectral difference between a treated and an untreated sample.

The results are expressed as 245 nm Difference Units (DU) per unit ofsample.

The method is very sensitive and specific for relatively purepreparations of xeronine. However, when it is used on crude enzymesolutions a variety of other materials also undergo a structural shiftwhich is not reversible by re-adjusting the pH. Some of these materialsabsorb close enough to 245 nm to affect the height of the peak.Nevertheless even with crude enzyme preparations the method has goodcomparative value between different batches of enzymes.

Casein Aggregation Method (CAU)

For routine laboratory work this is the most convenient method since itis rapid, many samples can be handled at the same time, it isinexpensive, and it is sensitive. This assay is based on my discoverythat xeronine can simulate certain of the reactions of rennet; xeroninecan liberate a peptide from casein which leads to aggregation of thecasein.

This method does have certain shortcomings. However, if the limitationsare recognized and handled, the method is reliable.

The principal interfering materials are proteases, excessive saltconcentration, certain divalent cations (especially calcium), andexcessive amounts of cysteine. The protease problem can be easilyhandled by placing the sample carefully in the bottom of a test tube andthen heating the entire test tube in a boiling water bath for 5 minutes.The salt problem can generally be handled by dilution. Ammonium acetate,the salt which will be most commonly encountered in xeronine samplesprepared by adsorption on weak cation columns, will interfere in theassay at concentrations down to 1/4 saturated solutions. Below thislevel they rarely cause a problem. Cysteine is occasionally a problemsince it must be added to certain steps in the preparation of xeronineto keep the precursor from forming disulfides. Adding a relativelyinsoluble mercury salt in excess, such as ethyl mercuric chloride, doesnot interfere in the reaction and quickly eliminates the excesscysteine.

The casein aggregation test may detect and assay both free xeronine andoccasionally proxeronine. Free xeronine reacts immediately with caseinto form an aggregation as soon as the solution is heated to 45 to 55 C.Proxeronine does not cause a reaction. However, certain batches ofcasein have as a contaminant an enzyme which, if other factors arepresent, can liberate xeronine from proxeronine. Heating the caseinsolution to 85-90 C. destroys this enzyme. If this enzyme is present incasein and if the sample contains proxeronine plus accessory factors,then the casein solution gradually becomes turbid over a period of anhour. This is definitely an enzyme reaction which can be readilyrecognized and distinguished from the rapid aggregation of casein causedby free xeronine.

In most of my studies I have purposefully prepared a crude caseinsolution from unpasteurized milk. This contains a maximum amount of boththe enzyme (proxeroninase) as well as part of the accessory factorsrequired for the enzymatic liberation of xeronine from proxeronine.Rather than using casein solutions which are designed to give aclassical clotting of the casein, I also purposefully use less calciumthan is normally recommended for casein aggregation reactions. Thisgives a solution which is generally clear enough to read in aspectrophotometer.

My standard casein aggregation substrate is a 1.5% casein solutioncontaining 0.001 M CaCl₂ adjusted to pH 6.0. This is made by suspendingthe casein in water, adding sodium hydroxide to raise the pH to 9,heating the suspension to 50 C. to aid in the solution of the casein,adding the stock calcium chloride solution, and then adjusting the pH to6.0 with a 2 M acetate buffer adjusted to 4.7.

For the assay four half dilutions of the sample are made and one ml ofeach is placed in a test tube. Four ml of the substrate are added toeach tube and the tubes are then placed in a 45 C. water bath for onehour. A similar set of dilutions is prepared but to each tube 0.2 ml ofa 0.1 M cysteine solutions is added before the substrate.

If the sample contains free xeronine and no prexeronine the "nocysteine" tubes quickly become turbid whereas the "+cysteine" tubes haveless aggregation and the specific amount of aggregation decreasesrapidly with dilution. If the sample contains proxeronine and no freexeronine, the "+ cysteine" tubes generally show much more aggregation.

The optical density of the sample tubes and the check are measured at aconvenient wavelength. I use 600 nm to avoid interference with colors incertain samples. After plotting the change in O.D. with concentration adecision is made about the possible components of the mixture. With somesamples a straight line can be drawn between the points. With others,because of the inhibiting effect of cysteine on the free xeroninereaction and its activating effect in promoting the liberation of freexeronine from proxeronine, the line drawn between the points on theconcentration curve may slope either downwards or upwards. This gives aclue about the reactions which have occurred. The CAU value is thencalculated:

    CAU/unit=(O.D. of sample-O.D. of check)×10

Blood Platelet Assay

This assay, as described by Heinicke et al. (Experientia 28, 844, 1972)is rapid, requires only a few drops of platelet rich plasma, and issensitive. Furthermore the assay has a direct relationship to one of thepotential applications of xeronine. The disadvantages are that plateletsfrom different people vary in their susceptibility to aggregation byadenosine diphosphate and that certain salts interfere in the reaction.

In the early stage of my research I used this method exclusively.

Smooth Muscle Contraction

This assay, using the smooth muscle from the stomach of a mouse, isexcellent for distinguishing between the action of xeronine andproxeronine. Xeronine gives an immediate response causing the muscle tocontract more intensely and increasing the frequency of the contraction.By contrast proxeronine gives exactly the same reactions but theresponse is delayed about thirty seconds and continues for about 30seconds after the bathing solution containing the proxeronine solutionis replaced with the standard salt solution.

Antiinflammatory Test

All critical samples were checked by a standard antiinflammatory test inwhich an irritant was injected into the skin of a mouse and then thetest substance was administered by i.p. injection. After an appropriatetime the mouse was killed and a uniform sized piece of skin removed witha punch made in the area of the injection. This was weighed from controland treated animals.

Disc Electrophoresis Assay

For complex mixtures, such as commercial enzymes, the fastest and thebest test, is a standard electrophoretic separation in acrylamide gelsin tubes. This is run by the standard Ornstein method at pH 4.5.However, at the completion of the run, the gels must be removed asrapidly from the tubes as possible and placed in the dye-fixativesolution. If this is not quickly done, the originally sharp band willquickly diffuse and become blurred and difficult to detect.

Also instead of using Coomassie Blue, the protein stain which mostbiochemists use today, Amido Black stain should be used instead.

Recovery of Xeronine from Natural Sources

The manipulations which must be used for the recovery of xeronine fromnatural sources are both complex and critical. Certain materials, suchas calcium must be present at a critical concentration. Too much or toolittle will lead to no formation of xeronine. Also certain materials,such as cysteine, act as necessary agents for one phase of the reactionand act as an inhibitor for another phase of the reaction. Thedirections which I have listed in this invention are specific in theirdescription of the reactions which occur at the molecular level butgeneral in their application to different raw material sources forxeronine recovery. However, a skilled biochemist will have no difficultyin applying the principles which I have enumerated to his specificproblem.

The reaction leading to the liberation of xeronine is deceptivelysimple:

    PX+PXase+accessory factors→X

where X=xeronine, PX=proxeronine, the precursor of xeronine and thesubstrate for the enzyme, and PXase is the enzyme which with theappropriate accessory factors leads to the formation of xeronine.However, each of the factors on the left hand side of the equation isitself involved in a series of reactions which greatly complicate thesimple reaction pictured above. Let us consider each of the factorsseparately.

Proxeronine (PX) is a moderately large, complex molecule containing nocarbohydrate moieties as measured by the anthrone test and containing noamino acids. The molecular weight appears to be in the vicinity of4,000. This molecular contains a free SH group which participates in theformation of mixed and homogeneous disulfide molecules. The disulfideform of PX (with one exception) is not active as a substrate for PXas e.Therefore in any raw material source for the extraction of xeronine, apreliminary estimate must be made of the possible status of theproxeronine molecules. If the solution contains large amounts ofglutathione, a common constituent of many fresh plant extracts, then nosulfhydryl reducing agents need be added to the solution. However, ifthe plant extract is old or if it has been exposed to air, then cysteineshould be added to the solution. Generally if commercial enzymepreparations, such as bromelain, pancreatin, or bacterial protease, areused as a source of recovering xeronine, the assumption can be made thatpart or all of the proxeronine will be in the disulfide form and thattherefore cysteine or a related reducing agent should be added to thesolution to reduce the disulfides.

Different proxeronine disulfides vary in their ease of reduction bycysteine or related reducing agents. The mixed disulfide containingproxeronine and glutathione is readily reduced by cysteine at pH 5-8 atroom temperature. By contrast the proxeronine-proxeronine disulfidemolecule is both extremely insoluble and is also difficult to reduce.This molecule will not dissolve in hot (90 C.) pH 11 solution nor willit dissolve in 0.05 M cysteine solution at pH 11. However, if the lattersolution is carefully and slowly warmed, the disulfide molecule will bereduced and will still serve as a substrate for proxeroninase. However,if the pH is higher than 11 or if the heating is prolonged longer thannecessary to reduce the molecule, then the molecule is destroyed.

Apparently the SH group of proxeronine is not involved in the reactionbetween the enzyme and the substrate. However, placing a bulky group onthe SH group of proxeronine, as occurs during most disulfide formation,blocks the proper positioning of the substrate and the enzyme andaccessory factors. Since the SH group is not directly involved in theenzyme action it should be possible to block the SH group of proxeroninewith a small group and thus prevent the formation of disulfides. If theblocking group is small enough, it will not interfere with the reactionof substrate and enzyme. Carrying out this blocking reaction bothincreases the stability and utility of preparations containingproxeronine and also increases the yield of xeronine during the stepslisted below. This is another critical part of the invention.

Many different agents can be used as blocking agents. The examples whichI am listing are not to be considered exclusive but merely asillustrative. In the laboratory I generally use a simple mercury saltsuch as ethyl mercuric chloride. This is convenient to use and is ableto keep the solutions saturated with trace amounts of mercury at allstages of the isolation steps. Another useful reagent is sodium sulfide.This has two actions. In the initial stage of the reaction it acts as areducing agent and aids in hydrolyzing disulfide bonds. However, at alater stage, it forms a mixed disulfide with proxeronine. However, sincethe added molecule is small, it causes no problem in the reaction of theenzyme with its substrate. At pH values above 5 iron salts form ablocking agent which is inexpensive and safe to use for food and medicalapplications. However, one of the best blocking agent is a thiosulfatemoiety. This can be formed on the SH group by the action oftetrathionate. (See Heinicke, U.S. Pat. No. 3,539,451).

The discovery of proxeronine and an elucidation of its role in theformation of xeronine is a discovery of major importance. Especiallyimportant was the demonstration that proxeronine contained a reactivesulfhydryl group and could readily participate in the formation of mixeddisulfide bonds. This discovery explained the confusing observationsthat at times cysteine was essential for biological activity whereas atother times cysteine actually inhibited biological activity. (Cysteineand other free sulfhydryl compounds interfere in the reaction ofxeronine and certain of its natural receptors.) The discovery of manymixed disufides containing proxeronine as one component explains themultiplicity of compounds which had distinctive UV absorption spectrabut which all eventually produced the same biological action.

To liberate xeronine from its substrate an active enzyme, which I havenamed proxeroninase, must be present. However, in most natural sourcesfor the recovery of xeronine, this enzyme does not exist in an activeform. Instead it exists as a very basic molecule which has anisoelectric point of about 10.5. (See Heinicke and Gortner EconomicBotany 11, 225-234, 1957.) A specific amidase probably hydrolyzes theamide groups of surface asparagine and glutamine groups on the proteinto convert the protein from a molecule having a very basic isoelectricpoint to one having an isoelectric point around pH 5.7. Then in thepresence of the proper concentration of free calcium ions the modifiedprotein molecules unite to form dimers or trimers. The demonstrationthat the basic molecules could form dimers with an isoelectric point ofabout 5.5 was done by Mr. Araki of Jintan Dolph. This work was doneindependently of the work of Klein and Houck who merely showed that theprotein which they isolated was a dimer or trimer.

My discovery that a critical concentration of free calcium ions isnecessary for the formation of proxeroninase is critical for thereliable formation of this enzyme. Since most substrates normallycontain enough calcium ions to act as inhibitors for the formation ofthe active enzyme, some method must be used to gradually reduce theconcentration of calcium ion. If this is carried out, then sometimeduring the reduction of the free calcium ion concentration theconcentration will be optimal for the formation of the di or trimermolecule.

Since most plant and animal tissues contain a variety of molecules whichcan chelate calcium ions, the simplest method of adjusting the calciumion concentration at this stage of our information about the compositionof animal and plant tissues is to carry out some manipulation which willreduce the calcium ion concentration gradually. Such methods are, butnot limited to, simple dilution, passage of a solution of a tissueextract over a cation exchange resin, dialysis, gel filtration, the slowaddition of either a calcium chelating agent or else the slow additionof a calcium precipitating agent such as oxalate or phosphate ions at pHvalues above 5.5 or sulfate ions at any pH. Some examples of uses ofthese techniques will be given in the examples.

Possibly one of the functions of certain natural chelating agents whichoccur in cells, such as citrate ions, uronic acids, heparin, as well ascertain large complex carbohydrate polymers which contain carboxylresidues is to provide a local concentration of calcium which is optimalfor the formation of PXase.

Based upon my preliminary suggested structure for proxeronine, I do notbelieve that the xeronine molecule exists preformed in the substrate.Instead I believe that proxeroninase is a mixed function enzyme whichsynthesizes the alkaloid by forming new covalent bonds and thenhydrolyzing others. The formation of new covalent bonds generallyrequires a source of energy which is normally supplied by ATP or by theNAD-NADH₂ system. Thus in addition to having the substrate (PX) and theenzyme (PXase) certain accessory factors are required. This is a problemwhich still requires additional work. However, I have found that eitherusing fresh tissue extracts or else adding extracts of yeast or othermicroorganisms to the mixture greatly improves the recovery of thealkaloid. I made this discovery when I found that solution of pineapplestem extracts which had been made from wet stems gave no recovery ofxeronine; however, if the solutions were passed over XAD-2 columns andthen left on the columns for several days before they were eluted, therecovery came back to normal. During this extended time on the columnbacterial growth occurred and probably supplied the critical factors. Isubsequently added either yeast or yeast extract to the juice to supplythe necessary accessory factors.

Both the discovery that a critical level of calcium ion concentration isrequired for the active enzyme (PXase) to be formed from the basicproteins and the discovery that certain accessory factors are necessaryto obtain good yields of alkaloid are two additional critical factorswhich will have application in pharmacology and in physiology. Twoadditional problems still remain to be solved; (1) the factors leadingto the activation of the amidases which convert the basic protein intoacidic proteins which in the presence of the proper concentration ofcalcium and the proper pH dimerize to form the active enzyme and thecomplete elucidation of the nature of the accessory factors which arenecessary for the reaction to occur. Nevertheless the information whichI am supplying in this patent is sufficient to enable chemists toreliably produce active preparations of xeronine from a wide range ofraw materials.

Once the free xeronine is formed and liberated, it generally is quicklyused or destroyed. Thus unless special precautions are taken, no freealkaloid will be obtained in spite of having all conditions necessaryfor the production of the xeronine. Part of the destruction of thexeronine is enzymatic and part is a simple oxidative destruction of themolecule. The enzymatic destruction can best be limited by adjusting thepH of the solution to a point where the enzyme is inactive. Thepreferred pH is on the acid side, namely pH 3.0 to 4.5 or 5.0. This lowpH also appears to lessen the oxidative destruction of xeronine. Anothermethod of eliminating the enzymatic destruction of xeronine is to heatthe solution to 65 C. for 15 minutes. This also has the advantage ofdestroying all of the proteases which--in spite of the presentpharmacological practices--are undesirable for most pharmacologicalapplications. Still another method of stabilizing the liberated xeronineis the formation of salts. Both organic and inorganic salts areeffective. With pineapple stem extracts one of the simplest salts torecover is one formed between xeronine and silicic acid. The pineapplestem juice is very high in soluble silicates. In the presence ofcations, including xeronine, the silicates can be precipitated byraising the pH above 6. Thus it is possible to both isolate xeronine asa silicate salt complex and also to stabilize the xeronine. Anothernatural salt complex which forms readily and can be isolated byprecipitation with acetone is the ferulic acid rich, complexcarbohydrate polymer which is found in pineapple stem juice. (Levand andHeinicke, Phytochemistry 7, 1659-1662,) 1968).

Isolation of Xeronine

The critical step in the isolation of xeronine is the liberation ofxeronine from the natural precursors. This subject was covered in theprevious section. Once the alkaloid has been liberated, then any of theclassical techniques of alkaloid chemistry are usable for isolation andpurification. These include, but are not limited to, partition betweenorganic and aqueous phases by changing the pH, adsorption on eitherstrong or weak cation exchange resins, adsorption on adsorbents such ascarbon or proteins by raising the pH above 6.5 to lower the solubilityof the alkaloid in water, by distillation, or by precipitating thealkaloid as an insoluble salt. Some examples of these techniques, butnot limited to them, will be given in the section on examples.

There are many feasible source materials suitable for the isolation ofxeronine and the present invention should not be construed as limited toany particular source materials.

Xeronine may be isolated from a wide variety of plant sources, includingtubers, plant latex, leaves, seeds, seed coatings and roots.Particularly rich sources are those plants which have a growth patternin which rapid growth periods alternate with long periods of quiescence.Some of these are the Bromeliaceae, the Ficus family, the Euphorbiaceae,the Caricaceae, some compositaceae, and many desert plants. Particularlypromising as raw materials are those plants which are already planted ingroves or plantations, such as rubber trees, pineapple, and agave andhennequin.

Xeronine may also be isolated from microbial sources. Here the criticalfactor is not so much the species of microorganism which is selected asis the growth cycle in which the organism is harvested.

Animals are also a potential source of xeronine. The richest sources areextracts of the stomach lining and pancreatic secretion.

Marine organisms are also potential sources of xeronine.

The potential level of xeronine isolated from said sources depends uponthe stage of growth of the cells or tissues, the best time being whenthe cells are in the resting or storage stage.

Instead of the original raw material, extracts of these tissues can alsobe used as a raw material for isolating xeronine. Some examples of suchextracts are "bromelain", papain, rennet, pancreatic amylase, fungalprotease and salmon milt.

Physiological Action of Xeronine

Xeronine's physiological action appears to be as a modifier of therigidity of specific proteins. Thus it can have a wide range of effectsdepending upon the function of the protein; it can convert certainspecific inactive proteins into active enzymes. For example the actionof xeronine with kappa casein converts this protein into an enzyme whichcan liberate by autodigestion a specific polypeptide. This actionexplains how xeronine coagulates casein. It may also activate the bodycollagenase, an action which could explain the unique ability of certainpreparations made from the pineapple plant to debride burn eschars by awell recognized collagenase action. It is the factor which convertscertain inactive plant and bacterial amylases into active amylase.

If the protein receptor occurs on a cell membrane, then the reaction ofxeronine with the receptor may affect the transfer of materials into thecell. The cell membrane receptors very likely require both a specifichormone as well as xeronine before a specific reaction occurs. This dualrequirement could explain why "bromelain" may simulate the action ofcertain hormones, such as the prostaglandins, insulin, adrenaline, themilk secreting hormone, and many other diverse types of hormones.

One of the outstanding properties of xeronine is its great physiologicalactivity even at extremely high dilutions. Based on its action inactivating the proenzymes of seed fragments of barley, I estimate thatit still shows biological activity at a dilution of 10⁻¹⁰ g of xeronineper gram of substrate. This makes biological assay of xeroninecontaining products easy to measure but makes chemical work with thismaterial very difficult because of the problems in isolating sufficientquantities of the material for conventional chemical analysis.

APPLICATIONS OF XERONINE

Pharmacological Application

Every pharmacological action of such "enzymes" as bromelain, bacterialproteases, and pancreatin may be ascribed, I believe, solely to apotential source of xeronine which these enzymes may contain.

This belief is based on the demonstration that samples of xeronineprepared by distillation, and which therefore could not possibly containpeptides, amino acids or steroids, acted as excellent antiinflammatoryagents when injected into mice, inhibited the in vitro aggregation ofblood platelets by adenosine diphosphate, caused the debridement of burneschars on mice, stimulated the partial breakdown of wheat grits, andcaused the aggregation of casein. All of these are reactions formerlyimputed to such proteases as "bromelain" pancreatin, and Serratiamarcescens protein. Therefore standardized preparations of xeronineshould be more effective for all of the present medical, food, andindustrial applications of bromelain and the other enzymes since theseare improperly standardized and therefore unreliable in performance.

However, there are several important new applications for xeronine. Mydemonstration that the active ingredient in many of thepharmacologically active enzymes and in many of the effective folkloredrugs is an alkaloid, namely xeronine, and that this alkaloid can berecovered from animal and bacterial sources, indicates that thisalkaloid is a critical normal metabolic coregulator. Therefore one wouldpredict that xeronine would be an effective antidote against alkaloidpoisoning and addiction. In the examples I have shown that a relativelypure sample of xeronine was an almost perfect antidote for tetrodotoxin,the most toxic alkaloid known. This confirmation of the theory led me tosuggest that xeronine should be a specific cure for nicotine and harddrug addiction and for alcoholism. We have tested crude preparation ofxeronine on confirmed smokers and have had a 90% cure rate with notension involved during the withdrawal period. With hard core drugaddicts, xeronine should provide a true cure with no withdrawalsymptoms. Preliminary tests with crude preparations of xeronine haveshown complete cures of hard core drug addicts with no withdrawalsymptoms and with no dependency on a substitute alkaloid.

Another critical potential application for xeronine is for thealleviation of the symptoms of one type of senility. This observationwas originally made by Gus Martin with a sample of enzyme which I hadprepared in the early phases of my research. This particular sample wasmade into pills and given to a woman who had been senile,uncomprehending, immobile, and incontinent for three months. Two hoursafter taking the pill she sat up in bed, asked why she was there andbegan asking for her family. As long as she was taking the pills, shewas a normal, functioning person again and took a very active part inthe hospital program. When the supply of this batch of pills ran out, an"improved" batch of bromelain pills was substituted. Three days latershe lapsed into her former senile state. Until my recent work onxeronine, I had been unable to repeat or to explain why this one batchof bromelain behaved so spectacularly.

Another important new application for xeronine will be as a generalstimulant or tonic. In the tetrodotoxin experiment mentioned above, thecontrol mice which had been injected with xeronine only, became veryalert and explored their cage for about a half an hour before they alsoburrowed into the shavings as the saline injected mice had doneimmediately after being injected. Based on this behaviour in mice Idrank a solution of xeronine containing about 50 times as much xeronineas the mice had received. The pleasant, stimulating, alert feelinglasted until about three o'clock in the morning. This is a responsewhich is similar to that reported by Russian scientists for extracts ofhigh quality ginseng or Eleuthrococcus.

In summary as far as the medical applications of xeronine are concernedmy discovery that xeronine can counteract the effects of foreignalkaloids will suggest many new and important applications in medicine.Also another discovery which will have important medical applications ismy finding that xeronine acts as the coregulator for many hormoneactions. This suggests that the body has a two component system forregulating and integrating the metabolism of different tissues;hormones, which are secreted into the blood stream and contact alltissues and xeronine, which is produced locally by the tissue anddetermines whether or not that tissue will respond to the presence ofthe hormone in the blood. Both factors must be present for a response tooccur. This theory suggests that many problems, such as diabetes, may becaused either by a lack of the hormone, insulin, or by the lack ofxeronine in the cell membrane at the local level. Both must be presentfor the cell to properly absorb and metabolize glucose.

In plants xeronine has another function in addition to a possible roleas a coregulator with secreted hormones. In the pineapple plant xeronineconverts certain precursors of catabolic enzymes into the active form.Thus the liberation of xeronine, through its action in forming activehydrolases, converts stored food material, such as starch, proteins, andorganic phosphorus compounds into soluble sugars, amino acids andphosphorus. These can be used either to produce new growth or tomobilize food for storage in seeds or tubers.

This action has great utility in modifying the properties of such foodseed materials as peas, corn, lima beans, beans, wheat, rice etc. intoproducts in which the starch is partially broken down into sugar, thusproducing a sweeter and more tender product. Such an action also hasgreat value in hastening the germination of seeds by stimulating theconversion of the stored food products into simple usable molecules.

Another food application of great utility is my discovery that xeroninecan simulate the action of commercial rennet. This discovery nowprovides the food technologist with a method of forming a milk curdwhich can be used as a basis for making cheeses. Since the action ofxeronine is limited solely to the clot forming action and since there isno secondary proteolytic effects to consider, the scientist now hascomplete control over the cheese manufacturing process.

The casein coagulating ability of xeronine can also be used to advantagein cheese manufacturing processes.

Xeronine can also be used to produce better flavored products fromtissues which are fermented to develop flavors, for example, cocoa,vanilla, coffee, beer and wine.

Xeronine can also be used as an in vivo or in vitro meat tenderizingagent.

EXAMPLES A. ISOLATION OF XERONINE BY VARIOUS TECHNIQUES FROM VARIOUS RAWMATERIALS

In the examples given below the assumption is made that eitherconditions have been arranged so that free xeronine has been liberatedfrom the precursors or else that the technique used will allow freexeronine to be released during the isolation process.

A1 BY ADSORPTION AND ELUTION FROM XAD-2

Ex. 1 From Commercial Grade Bromelain.

An XAD-2 column was thoroughly cleaned and then rinsed with dilute HClfollowed by water to leave a slightly acidic reaction on the column.Twenty grams of commercial grade bromelain was suspended in one liter ofwater and then mixed with one liter of acetone. The solution wascentrifuged, the precipitate discarded and the supernatant solutionslowly run through the column. The percolate, which consisted of amixture of proteins, peptides, sugars, salts and simple organic acidswhich were soluble in 50% acetone was discarded. After the column wasthoroughly washed with a 50% acetone solution, the material adsorbed onthe column was eluted with one liter of a 50% acetone solutioncontaining sufficient ammonium hydroxide solution to raise the pH to10.5.

The eluate, which was straw colored and had a pH of 9, was adjusted topH 4.5 and the acetone stripped off under a vacuum. The insolubles whichformed during this stripping step consisted of denatured protein andproxeronineine residue and were discarded. The clear supernatantsolution was then run over a freshly cleaned XAD-2 column, the percolatecollected, and the solution concentrated to 15 ml. At this concentrationammonium salts crystallized out. These were discarded. The clearsupernatant solution was then freeze dried. Because of the high saltconcentration, this took several days.

Weight recovered (mostly ammonium salts) 12.5 g

Xeronine units per mg=1.8 CAU or 22,500 CAU total/20 g

The salt prepared from this and similar runs was further purified by thetechniques described in Section B of the Examples.

Ex. 2. From Commercial Rennet Powder

The excellent recovery of xeronine by the emulsion technique (Ex. 6)indicated that additional tests were warranted.

Fifteen grams of commercial rennet powder (Nakari) were suspended in oneliter of water. Because of the strong odor, the pH was dropped to 3.5and the simple organic acids extracted with chloroform. An equal volumeof acetone was added to the aqueous portion, the mixture centrifuged,and the precipitate discarded.

The acetone supernatant solution was then run over a freshly washedXAD-2 column, the column rinsed with 50% acetone and the adsorbedmaterial then eluted with one liter of 50% acetone which had beenadjusted to pH 10.8 with ammonium hydroxide solution.

The pH of the eluate was then adjusted to pH 5.0 and the acetone and thewater removed under a high vacuum. To the dry salts 10 ml of a pH 10 1 Mborate solution was added and the solution distilled under a highvacuum.

The distillate had a strong nicotine-like odor. The pH was adjusted topH 7.0 and the solution evaluated an an antiinflammatory agent for theprevention of edema in mice. It gave better protection against edemathan the standard test solution of bromelain which was used in thesetests.

A2. BY PASSAGE OVER COMBINATIONS OF ION EXCHANGE RESINS

Ex. 3. The Acetone Still Aqueous Residues from a Commercial BromelainProduction Plant

Five gallons of the acetone still residues were passed in series over astrong cation resin (C-20) in the hydrogen form, over a macroreticularnon-ionic resin, and over a weak cation resin (IRC-84) in the ammoniumsalt form. The final percolate solution was discarded.

The weak cation column was eluted with 5% acetic acid and those eluatessaved which had a pH between 7 and 4.5. The solution was concentrated ona thin film evaporator under a high vacuum. As the ammonium acetatesalts began to crystallize, they were removed and discarded. The thicksyrupy residue was then freeze dried. During the freeze drying stepadditional ammonium acetate distilled from the freezing flask.

    ______________________________________                                        Total weight of ammonium acetate + xeronine                                                             77.45 g                                             Average CAU/mg            5                                                   Total recovery from one gallon                                                                          77,450 CAU                                          ______________________________________                                    

Of about 15 runs by this method, this recovery was considerably abovethe average. During this isolation the adsorbed material remained on thecolumn for three days before it was eluted. This long holding step,under conditions which were favorable for the release of free xeroninefrom the precursors, is probably responsible for the excellent recoveryof xeronine.

A UV spectrum of this product showed a single sharp peak at 253 nm and asmall peak at 215. This particular batch of xeronine was used in many ofthe tests of possible applications of xeronine.

Ex. 4. Recovery of Xeronine from Bromelain by Combined Ion ExchangeResins

To a solution of 5 grams of bromelain adjusted to pH 3.8, an equalvolume of acetone was added and the precipitate was removed anddiscarded. After the acetone was removed from the supernatant solutionunder a vacuum, the aqueous residue was run over a strong cation resinin the acid form (C-20) and then over a weak anion resin (IRA-84) andfinally over a weak cation resin (IRC-50) in the ammonium salt form.

The weak cation resin was eluted with 5% acetic acid, the solutionconcentrated, part of the ammonium acetate removed as crystals and theresidue freeze dried. During the freeze drying operation, additionalammonium acetate was removed as a volatile salt.

    ______________________________________                                        Weight of recovered salt/5 g bromelain                                                                2.8 g                                                 CAU/mg                  4.3                                                   Total CAU recovered /g enzyme                                                                         2408                                                  ______________________________________                                    

Ex. 5. From Pancreatic Amylase

Seventy five grams of hog pancreatic amylase were dispersed in 850 ml ofwater containing 2 g of ascorbic acid. The pH was adjusted to 4.5 andthe insolubles removed. To the supernatant solution an equal volume ofacetone was added, the precipitate removed, and the acetone evaporatedin a thin film evaporator. During the evaporation of the acetone aprecipitate formed. This was removed. (Later studies indicated that thisprecipitate should have been saved as a source of proxeronine.) Theclear solution was first run over a strong cation column (C-20) in theacid form, then over a non-ionic macroreticular resin (XAD-2) andfinally over a weak cation in the ammonium salt form (IRC-84).

The IRC-84 column was rinsed first with water and then with 100%acetone. (Later studies showed that some xeronine could have beenremoved by this last step). The column was then eluted with 5% aceticacid. Xeronine activity started to appear in the eluate when the pH ofthe column had dropped to 6. Elution was continued until all of thecolumn was in the hydrogn form.

    ______________________________________                                        First portion of IRC-84                                                                        30 g salt  0.22 CAU/mg                                       eluate after pH 6.0                                                           Next portion     32 g salt  0.09 CAU/mg                                       Last portion     28 g salt  0.0 CAU/mg                                        ______________________________________                                    

The recovery of CAU was 126.4 CAU/g of amylase. Whereas this is not ashigh as from other sources, this is acceptable considering thatpotential activity was lost in two fractions, the precipitate whichappeared when the acetone was removed from the first supernatantsolution and the acetone wash of the IRC-84 column.

A.3 BY THE EMULSION TECHNIQUE

Ex. 6. From Commercial Rennet Powder

Four grams of commercial rennet powder (Nakarai) were dispersed in 150ml of water and the pH adjusted to 5.7. After an hour the pH was raisedto 10 and the solution was vigorously shaken with and extracted with two50 ml portions of chloroform. After centrifuging the similar fractionswere combined. The tight emulsion layer was broken by adding fivevolumes of methanol and then centrifuging the suspension to remove theprecipitated proteins. The solvents were removed from all samples andthe solutions assayed.

    ______________________________________                                        Aqueous phase        0                                                        Emulsion precipitate 0                                                        Emulsion supernatant solution                                                                      83,480                                                   Clear chloroform layer                                                                             trace                                                    ______________________________________                                    

Ex. 7. From Commercial Bromelain

Five hundred ml of a 2% bromelain solution were mixed with sufficientcysteine to give a 0.01 M solution and the pH then adjusted to 8.5.Twice the solution was vigorously shaken with 50 ml of chloroform in aseparatory funnel to promote the formation of a tight emulsion. Thesolutions were centrifuged and the similar fractions combined. The clearchloroform layer was extracted twice with 50 ml of pH 3 buffer and theaqueous phase used for assay. The emulsion layer was mixed with fourvolumes of acetone and the mixture centrifuged to remove theprecipitated colloids. The solvent was removed from the supernatantsolution and the aqueous residue assayed.

    ______________________________________                                                  Not Boiled                                                                              Boiled                                                    ______________________________________                                        Aqueous layer                                                                             (Protease)  0.2 CAU/ml 100 total                                  Extract of   14 CAU total                                                                             0.3 CAU/ml  12 total                                  chloroform layer                                                              Emulsion layer                                                                            160 CAU total                                                                             12 CAU/ml  150 total                                  ______________________________________                                    

The recovery of xeronine in this experiment was low compared to therecovery on XAD-2 columns or on ion exchange resin columns. Thedifference could be partly a time factor. In the emulsion technique usedin this example, the run was completed in about 1/2 hour. By contrast inany of the column techniques, a run generally takes from 5 to 48 hours.This longer time permits more opportunity for the enzymatic formation ofxeronine. Also in the short run used in this emulsion example, thesample was at an unfavorable pH for enzymatic action most of the time.

The recovery of activity in this example should be compared with theexcellent recovery from rennet powder (Ex. 6) or from ficin (Ex. 8)using a similar technique but including a holding step at pH 5 of onehour to promote the liberation of xeronine.

Ex. 8. Recovery of Xeronine from Ficin by the Emulsion Technique

Ten grams of commercial grade ficin were suspended in 200 ml of waterand the pH adjusted from 5.0 to 3.5. The organic acids were thenextracted from this solution with chloroform. The pH of the solution wasthen raised to 5 and the solution held for a half an hour for thepossible liberation of free xeronine from the precursors.

The pH of the solution was then raised to 9.7 and the solution shakenvigorously with 50 ml of chloroform to form an emulsion. The mixture wascentrifuged to break the weak emulsions and to give a three phasesolution, a very tight, stable emulsion layer, and a clear chloroformlayer. The chloroform layer was extracted with pH 3 aqueous buffer toextract any bases from the chloroform layer. The emulsion layer wasbroken and the colloids precipitated by adding four volumes of methanolto the solution and centrifuging. Both the aqueous supernatant solutionsand the two fractions from the emulsion layer were boiled beforeassaying for CAU activity.

    ______________________________________                                        Assayed for CAU                                                                              No cysteine                                                                             Added cysteine                                       ______________________________________                                        Aqueous supernatant solution                                                                   2/ml   400 total                                                                              4/ml 800 total                               Emulsion layer; precipitate                                                                    0      0        0    0                                       Emulsion layer; supernatant                                                                    29.1   14,560"  52.5 26,260"                                 ______________________________________                                    

The recovery of 2626 CAU/g of enzyme compares favorably with therecovery from bromelain by the better techniques.

Note that in this example the recovery of xeronine was much higher thanin Example 8 with bromelain. In that example no opportunity was allowedfor the enzymatic liberation of xeronine before the extractiontechnique.

A6. BY GEL FILTRATION

Ex. 9. From Commercial Bromelain

This example is given not as a potential commercial method for producingxeronine but as an illustration of the liberation of xeronine fromcolloids in bromelain. The experiment described in this example was runtwenty times at different pH adjustments of the gel filtration columnand of the enzyme solution.

To summarize the results, I found the best recovery of xeronine when thecolumn and the enzyme solutions were adjusted to pH values between 4-5.Below pH 3.5 the recovery of xeronine dropped to negligibly low values.Between pH 6 to about 8 the recovery was 0. From pH 8.5 to 10.5 therecovery of xeronine was present but only about a third as large as atpH 4-5. I have illustrated below one such a test.

After equilibrating a G-15 Sephadex column with the desired buffer, Iadded 5 ml of a 1% solution of bromelain to the column and developed thecolumn with buffer. All tubes were assayed for protease activity, forCAU, for pH and for UV spectra.

With the column and the collection volume which I used, the proteaseappeared in tube 8, peaked at tube 9, and passed out of the column bytube 11. The first material to emerge from the column was a large,complex aggregate. This contained large amounts of an acidiccarbohydrate polymer, small amounts of a non-proteolytic enzyme, acidphosphatase, peroxidase, and lipophyllic materials. This aggregateconsistently appeared in tube 6, peaked at tube 7, and trailed into tube8. Xeronine was liberated from this colloidal complex and generallyappeared in tube 7. Some of the liberation apparently occurred as thecolloids were migrating down the column. This caused a tailing of thexeronine activity across all of the tubes. Much of the xeronine wasliberated in the collection tubes since an immediate assay of theactivity was frequently less than one tenth the value found after sixhours.

When the tubes containing xeronine from different runs, that is tubes6-7, were combined, concentrated, and then rerun on the column, thexeronine activity now appeared in tubes 15-18. These experimentsconclusively demonstrate that xeronine is liberated from materials whichare present in bromelain.

B. PURIFICATION

B1. BY GEL FILTRATION

Ex. 10. Of salts from an XAD-2 Isolation Procedure

500 mg of the preparation from example #1 were dissolved in three ml ofwater and placed on a Sephadex G-15 gel filtration and developed withwater. Ten ml fractions were collected. (See the FIGURE). All fractionswere checked for pH, for CAU and for the UV spectrum. All of theactivity appeared in essentially one tube, #17. This gave a spectrumwhich indicated that the fractions were still a mixture of peptides andxeronine. The UV absorption curve indicated that part of the absorptionwas caused by the 253 nm form of xeronine.

B2. BY DISTILLATION

Ex. 11. Of Salts from an XAD-2 Isolation Procedure of Bromelain

Five grams of the salt from Example 1 were dissolved in 50 ml of water,the pH raised to 9.5 and the solution distilled under a high vacuum witha dry-ice acetone cooled receiving flask. A mixture of ammonia vapors,water and xeronine distilled over and froze in the flask. When thefrozen mass was melted about five micrograms of xeronine crystalsfloated on the surface of the basic solution.

Xeronine crystals prepared by this method were used for mass spectralanalysis, for biochemical assays, for pharmacological assays, and forphysiological studies.

Although xeronine prepared by this method is of excellent purity, therecovery is low, about 1 to 2%. Apparently at the high pH used for thedistillation, a large portion of the xeronine is destroyed. Whatdistilled over had the typical nicotine-like odor which ischaracteristic of good samples of xeronine. This odor is so distinctive,that xeronine can be recognized on the basis of the odor alone.

Ex. 12. Protecting the SH Group of Proxeronine with Iron and Ascorbate

This example illustrates the use of ferrous sulfate and ascorbatecombinations to lessen the potential oxidation of the SH group ofproxeronine to form disulfides which are inactive.

Four 100 ml solutions of 1% bromelain were mixed with variouscombinations of iron and ascorbate at pH 8. After one hour at roomtemperature 150 ml of acetone was added to each beaker and theprecipitated colloids were removed. The pH of the supernatant solutionwas dropped to 5 and the acetone removed on a thin film evaporator. Allsolutions were boiled to destroy any residual protease activity, the pHraised to 10 and each solution shaken vigorously with 50 ml ofchloroform. Assays for casein aggregating activity were run on both theaqueous layer and on an acid extract of the chloroform layer with thefollowing results.

    ______________________________________                                        Treatment per 100 ml                                                                             CAU Activity in                                            ml 20%    g            Aqueous  CHCL.sub.3                                    FESO.sub.4                                                                              ascorbate    layer    layer                                         ______________________________________                                        0         0             57      30                                            0         1            300      56                                            4         0            320      53                                            4         1            280      56                                            ______________________________________                                    

Experiment 13. Recovery of Xeronine from Poor Quality Acetone StillResidue

This particular batch of acetone still residue came from a batch of wetstumps which had been processed for bromelain. A UV spectrum analysis ofthe solution showed a total lack of any material absorbing at 275 nm.This peak is a UV indicator of the quality of the raw material. Whenthis batch of solution was processed by exactly the same technique usedin Example 3 on the same columns, the recovery of xeronine from anycolumn was negligible.

This poor batch of juice was concentrated to 1/4 volume and the solutionwas set aside in the refrigerator for several weeks. During this time amold grew on the surface of the solution. This was removed and the juiceprocessed by passing one liter of the concentrated solution over anXAD-2 column which had been rinsed with dilute acid. This treatmentremoved most of the colored material.

In contrast to the behaviour of solutions of bromelain-acetonesupernatants, as in Example 1, in this run all of the CAU activityappeared in the initial percolate solution from the column. None of theeluate solutions contained any activity. This indicates that thereactions liberating free xeronine had occurred in this poor batch ofacetone supernatant solution during the several week holding period.Presumably accessory factors supplied by mold as well as a gradualprecipitation of calcium salts which occurred during this long holdingperiod, allowed the basic proteins to form active proxeroninase. Thistogether with the accessory factors led to the formation of freexeronine.

Before the several week incubation period and the addition of accessoryfactors through the growth of molds, the total recovery was about 5,000CAU/gallon. After the treatment described above the recovery was 64,000CAU/gallon which compares favorably with the best of the recoveries fromthis source.

This is a critical discovery.

D. APPLICATIONS

Ex. 14. Lessening of Blood Clot Formation on Intravenously InsertedCatheters

The Amplatz method (Durst, S; Leslie, J; Moore, R; and Amplatz, K.Radiology 1974;599-600) was used to form and quantitate clot formation.

Before a 30 cm length of catheter was inserted into the femoral arteryof 25-30 kg dogs, a 7 CAU/ml solution of xeronine in saline was pumpedinto the forepaw vein at the rate of 50 ml per hour. The injection wascontinued as long as the catheter was kept in the vein. After an hourthe catheter was removed, and the adhering clot weighed.

Immediately after removing the catheter and after discontinuing theinjection of xeronine into the animal, a fresh catheter was placed inthe artery and left in the animal for an hour. It too was then removedand the adhering clot, blotted and weighed.

    ______________________________________                                        Weight of clot before injection                                                                          309 mg                                             Weight of clot during xeronine injection                                                                 158 mg                                             Weight of clot one hour after discontinuing                                                              299 mg                                             injection                                                                     ______________________________________                                    

In another experiment enteric coated bromelain granules were fed to theanimal one hour before the catheters were inserted. The catheters werethen placed in the femoral artery every hour, removed, and the weight ofthe adhering clot weighed.

    ______________________________________                                        Weight of Adhering Clot at Different Intervals After an Oral                  Dose of Bromelain                                                             One Hour                                                                              Two Hours Three Hours                                                                              Four Hours                                                                            Five Hours                               ______________________________________                                        380 mg  142 mg    150 mg     205 mg  287 mg                                   ______________________________________                                    

The advantage of the xeronine injection technique over giving apotential source of xeronine orally is that the effect on the rate ofblood clot formation is immediate and continues only as long as theinjection is continued. This technique would have great utility inmedicine over the standard heparin technique.

Ex. 15. Effect of Xeronine on in vivo Meat Tenderization

One ml of physiological saline containing either 0, 4 or 10 CAU ofxeronine or a highly purified sample of bromelain at the rate of 20mg/kg was injected into the wing veins of four 18 month old cocks. Noneof the samples caused any visible distress to the chickens. Five minutesafter the injection the chicken were killed and dressed on a commercialprocessing line.

The chickens were cooked in a restaurant style oven by a professionalcook and coded samples of the breast meat presented to four judges. Nodetectable differences were noted in the flavor of the chickens. Therewas marked difference in the tenderness.

    ______________________________________                                        Ranking of the Tenderness of Meat on a 1-5 Scale                              1 = very tough, 5 = very tender                                               ______________________________________                                         0 CAU/ chicken  2, 3, 1, 2                                                    4 CAU/ chicken  3, 2, 2, 3                                                   10 CAU/ chicken  3, 5, 5, 4                                                   Purified bromelain                                                                             1, 1, 2, 1                                                   ______________________________________                                    

The bromelain sample had been purified by ammonium sulfateprecipitation, dialysis, and gel filtration. It contained 3200 GDU/g incontrast to the standard 1200 GDU/g for standard bromelain. What issurprising is that this sample caused no visible distress to thechicken. Normally commecial grade bromelain has an L.D.-50 of 15 mg/kg.Also this excellent protease sample actually increased the toughness ofthe meat.

The xeronine sample contained no trace of protein or polypeptides.Therefore the tenderizing action must be solely attributable to theaction of xeronine in activating the cathepsin hydrolases in the chickenmuscle.

Ex. 16. Hydrolysis of Seed Starch

Xeronine hydrolyses seed starch by activating the proamylases containedin the seed.

To 10 g of ground barley seed either 1 ml of buffer or 1 ml of buffercontaining 2 CAU of xeronine (prepared by the method shown in Ex. 3)were added to the mixture held at 40 C. for one hour. One hundred ml ofwater were then added to each sample and the suspensions placed in aboiling water bath for exactly one minute. The mixture was thencentrifuged and the supernatant solution decanted.

    ______________________________________                                                  ml supernatant                                                                           Viscosity of                                                       solution   solution                                                 ______________________________________                                        Control     51           similar to water                                     Xeronine treated                                                                          43           so viscous that it                                                            was difficult to pour                                ______________________________________                                    

This example illustrates the powerful action of xeronine in convertingone form of stored food, the starch, to soluble forms through theactivation of endogenous enzymes.

Ex. 17, Action on Casein

This example illustrates the ability of xeronine to liberate a peptidefrom casein similar to the action of the so-called "milk clotting"enzymes. Since this sample of xeronine had been boiled, there is nopossibility that the action could have come from contaminatingproteases. The xeronine sample was prepared as in Example 3.

To 100 ml of a 1% Hammarsten casein solution were added 25 CAU ofxeronine. Five ml samples were removed from the solution (at roomtemperature) at the times indicated, and mixed with 5 ml a precipitant.The precipitant contained 6% trichloracetic acid, sufficient acetate togive a molarity of 0.2 M and a pH of 4.8. The precipitate was removed byfiltration through a fine grade filter paper. To 2 ml of the filtratewere added 2 ml of a 4% NaOH solution and 1 ml of biuret reagent. Thebiuret color was read at 545 nm against a reagent blank.

    ______________________________________                                                       Time of Sampling                                                              0   4     90     150  170  210                                 ______________________________________                                        Net increase in biuret color                                                                   0     5     20    60   40   40                               ______________________________________                                    

That this reaction reaches a plateau indicates a reaction limited by theavailability of additional substrate. This type of reaction is verydifferent from that found with a standard protease, such as bromelain,in which the liberation of peptides continues for many hours.

Ex. 18. Action on Milk

A sample of xeronine prepared by distillation (Example 11) was added tocommercial skim milk and the mixture held at 45 C. A typical clot formedwhich showed syneresis within one hour.

Since this sample of xeronine could not possibly contain proteasecontaminants because of the method of preparation, and since this samplesimulates the action of commercial rennet solutions on casein, thisexample illustrates the utility of xeronine for cheese manufacture.

Ex. 19. Effect of Xeronine on Bleeding Time

This action can only be detected in animals or people who have beenstressed. In this experiment adrenaline was used as a stressing agent.

    ______________________________________                                        The Effects of Xeronine on the Bleeding Time                                  of Mice Which Had Been Injected with                                          Adrenaline One Hour Previously                                                Treatment                                                                              Time   Bleeding Significant Differences                              Code Time 0    60 Min.  Time   AX   BX   AB   BB                              ______________________________________                                        AX   Adrenaline                                                                              Xeronine 202.7  --   --   --   --                              BX   Buffer    Xeronine 150.7  --   --   --   --                              AB   Adrenaline                                                                              Buffer   129.1  **   no   --   --                              BB   Buffer    Buffer   122.6  **   no   no   --                              ______________________________________                                    

The mice were bled five minutes after the xeronine injection. Thevolumes used for i.p. injection were all 0.25 ml. The results wereanalyzed statistically with the odds shown. The * denotes differencessignificant at the 5% level; the ** denotes differences significant atthe 1% level. The amount of adrenaline used was 0.1 mg per kg.

Ex. 20. Effect of Xeronine on Inflammation

The rats were fasted overnight and then given 0.7 ml of either asolution of xeronine in 0.005 M cysteine or else 0.7 ml of physiologicalsaline containing 0.005 M. cysteine. One hour later each rat wasinjected subcutaneously with 0.05 ml of an anti-rat serum. Two hourslater the rats were killed and the extent of the edema was measured bypunching out a piece of skin at the site of injection and weighing thepunched skin.

The results are statistically significant.

    ______________________________________                                        The Effect of Orally-Administered Xeronine                                    on Serum-Induced Inflammation                                                 as Measured by the Punch Method                                                        Control                                                                              Treated                                                       ______________________________________                                                   117      86.1                                                                 133      115                                                                  117      110                                                                  127      77.7                                                                                       "t" test                                                133      115                                                                                        t.sub.o = 2.86                               Sum        627.3    503.8                                                                                      t.sub..05 = 2.31                             Average    125.5    100.8                                                     ______________________________________                                    

MISCELLANEOUS

Example 21 Analysis of Complex Samples for Free Xeronine

Up to about 1964 the electrophoretic patterns run at pH 4.5 of allbromelain samples prepared by Dole contained a thin, fast moving bandwhich stained blue with Amido Black stain. Since about 1966 none of theDole samples nor none of the Taiwan samples contained this fast movingband. This fast moving band represents free xeronine.

The sample was prepared by the method used in Example 3. When applied toa disc electrophoresis column and run at pH 4.5, a 5 mg/ml concentrationgave no visible bands. However, at 50 mg/ml a thin fast moving band,which was easily visible without staining, moved down the column. Duringthe interval between removing the sample gels from the tubes andstaining them with Coomassie Blue, about 20 minutes, the band spreadappreciably, indicating a small molecule. The dye solution in the tubecontained an appreciable amount of precipitate indicating that a fastdiffusing molecule moved out of the gel and reacted with the stainfaster than the stain moved into the gel.

This band, which moved twice as fast as the fastest protein band inbromelain (which has an isoelectric point of about 9.5), was cut out andtested for both its casein aggregating properties and also for itsability to inhibit the aggregation of blood platelets which had beenexposed to different concentrations of adenosine diphosphate. Itspositive action in both tests indicates that the band containedxeronine.

Of the two dyes, Coomassie Blue and Naphthol Black, normally used asprotein stains in disc electrophoresis, the latter, with a molecularweight of 616 is superior to the former, M.W. 854. With naphthol blackthe spread of the band and the loss of material by diffusion into thetube is less since the diffusion rates of the dye and xeronine are moresimilar.

Although the invention has been described with reference to specificembodiments, the exact nature and scope of the invention is defined inthe following claims.

I claim:
 1. A new composition of matter called xeronine formed by theprocess comprising obtaining source materials selected from the groupconsisting of plant, bacteria and animal alkaloid producing lipophilicextracts, combining lysozymes with the extracts to produce a mixture,controlling the concentration level of free calcium ions in the mixtureto produce an optimum concentration level which activates the lysozymes,and controlling the pH of the mixture to the range of about 3.5-5.0 toreact the extracts with the activated lysozymes to produce the activesubstance xeronine.
 2. The composition of claim 1 wherein the xeronineis an alkaloid having an essentially flat UV spectrum which develops anirreversible UV absorption peak at 245 nm when heated at strongly basicpH values and reversible UV absorption peaks at 253 nm and 280 nm whenheated at moderately basic and acidic pH values respectively.
 3. Thecomposition of claim 1 wherein the lysozymes are contained in compoundsselected from the group consisting of bromelain, ficin, papain and eggwhite.
 4. The composition of claim 1 wherein the lysozyme is a basicprotein molecule having an isoelectric point of about 10.5.
 5. Thecomposition of claim 1 wherein the optimal concentration of free calciumions is provided by gradually reducing the concentration of calciumions.
 6. The composition of claim 5 wherein the gradual reduction isachieved by a process selected from the group consisting of simpledilution, passage of a solution of a tissue extract over a cationexchange resin, dialysis, gel filtration, slow addition of a calciumchelating agent, and slow addition of a calcium precipitating agent. 7.The composition of claim 5 wherein the chelating agents are selectedfrom the group consisting of citrate ions, uronic acids, heparin andcarbohydrate polymers containing carboxyl residues.
 8. The compositionof claim 5 wherein the percipitating agent is selected from the groupconsisting of oxalate or phosphate ions at pH values above 5.5 andsulfate ions at any pH values.
 9. The composition of claim 1 furthercomprising isolating the xeronine.
 10. The composition of claim 9wherein isolating is carried out by a process selected from the groupconsisting of partition between organic and aqueous phases by changingpH, adsorption on either strong or weak cation exchange resins,adsorption on adsorbents such as carbon or proteins by raising the pHabove 6.5 to lower solubility of xeronine in water, distillation andprecipitation of xeronine as an insoluble salt.
 11. The composition ofclaim 9 wherein isolating is carried out by extracting xeronine fromaqueous solution into organic solvent at pH values above 7.8 andextracting the organic layer with weak acid solution.
 12. Thecomposition of claim 1 wherein the plants have a growth pattern in whichrapid growth periods altenate with long periods of quiescence.
 13. Thecomposition of claim 1 wherein the plants are selected from the groupconsisting of the Bromeliaceae, the Ficus family, the Euphorbiaceae, theCaricaceae, the Compositaceae and desert plants.
 14. The composition ofclaim 1 wherein the plants are selected from the group consisting ofrubber trees, pineapple, agave and hennequin.
 15. The composition ofclaim 1 wherein the animal material is selected from the groupconsisting of stomach lining and pancreatic secretion.
 16. Thecomposition of claim 1 wherein the extracts are selected from the groupconsisting of bromelain, papain, rennet, pancreatic amylase, fungalprotease and salmon milt.
 17. The composition of claim 1 wherein theplants are selected from the group consisting of tubers, plant latex,leaves, seeds, seed coatings and roots.